The protective antigen protein (PA) of B. anthracis is the key immunogen in currently approved and candidate vaccines designed to protect humans from infection. The present study, completed in 2018, examined the effect of PA expression on the metabolism of an optimized B. anthracis production strain, BH500. RNA-seq analysis was used to compare a strain expressing recombinant PA (rPA) from a shuttle plasmid with a control strain having an empty plasmid. The strains were grown to high density in a bioreactor under conditions yielding up to 180 mg PA per liter. The strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase P54P, sigB, the general stress transcription factor gene, its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There was decreased expression of several heat stress related genes, including groL, groES, hslO, narJ, hscC, dnaJ, dnaK, grpE, clpB, and clpP. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production. In this reporting year, we also worked to further improve the BH500 strain by identification, deletion and mutation of the HtrA protease. Proteases related to HtrA are widely distributed in prokaryotes and eukaryotes, where they play key roles in protein secretion. HtrA serves as both a chaperone aiding protein folding and as a protease that degrades proteins that fail to fold correctly. Deletion of the B. anthracis HtrA was performed with the IntXO-PSL system, yielding strain BH510, deficient in eleven proteases. An auto-processed and secreted form of HtrA was purified to homogeneity and used to produce antibodies to this protease. A mutation in the protease active-site of HtrA prevented its auto-processing, leaving HtrA in a cell-associated form. The BH500 and BH510 strains were compared for the ability to produce several anthrax toxin variant proteins which are being developed as tumor-targeting agents. In particular, we have characterized the expression, secretion, and integrity of proteins containing the N-terminal domain of lethal factor (LFn) fused to the cytolethal distending toxin subunit B (CdtB), the YopJ protein from Yersinia pestis, and the Mycobacterium tuberculosis ESAT-6 protein. These comparisons confirmed that HtrA has the dual roles of being both a folding chaperone and a quality control protease. The third project conducted during 2018 concerned the AtxA regulator of virulence factor expression in B. anthracis. Expression of the key virulence factors, specifically the anthrax toxin proteins and the anti-phagocytic capsule, are tightly regulated, being expressed only in the presence of carbon dioxide, which is a signal that the bacteria are within a mammalian host. AtxA is required for this response. We previously characterized the function of AtxA using Next Generation sequencing and found a positive correlation between the atxA copy number and the expression level of the pagA gene encoding B. anthracis protective antigen. In 2018, using electrophoretic mobility shift assays we obtained the first direct evidence that AtxA binds specifically to the promoter of pagA, upstream of the RNA polymerase binding site. We also demonstrated that in vitro CO2 appears to have no role in AtxA binding to DNA, but phosphomimetic or ablative mutations in the PRD domains do alter AtxA binding. DNase I footprinting, and in vitro analyses provided evidence that a stem loop in the pagA promoter is important for AtxA binding in vitro. Our study clarifies the mechanism by which AtxA interacts with one of its targets.